Mechanical studies of single ribosome/mRNA complexes

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• Investigating the mechanical properties of individual biomolecules like RNA and protein complexes is crucial for understanding their function.
• Ribosomes, the cellular machinery for protein synthesis, are complex molecular machines whose mechanical behavior under force is not fully understood.
• Previous methods lacked the precision to measure the mechanical response of ribosome-RNA complexes at the single-molecule level.

Purpose of the Study:

• To develop a novel methodology for immobilizing Escherichia coli 70S ribosomes on a surface for single-molecule mechanical studies.
• To investigate the force-extension relationship and mechanical properties of individual ribosome-polyuridylic acid (poly(U)) complexes.
• To determine the effect of tRNA binding on the mechanical stability of ribosome-mRNA complexes.

Main Methods:

• Developed a cysteine-reactive glass surface for specific anchoring of Escherichia coli 70S ribosomes.
• Utilized laser tweezers to apply force and measure the extension of individual ribosome-poly(U) complexes.
• Employed streptavidin-coated microspheres to bind biotinylated poly(U) templates of varying lengths.
• Implemented a novel optical method for precise control of the laser trap position (2 nm resolution).

Main Results:

• Successfully immobilized active Escherichia coli 70S ribosomes capable of binding poly(U) templates.
• Measured the force-extension relationship of poly(U) within ribosome complexes, revealing persistence length and backbone elasticity.
• Observed that some immobilized ribosome-poly(U) complexes could withstand forces up to 100 pN.
• Demonstrated that binding of an initiator tRNA analog (N-acetylated Phe-tRNA(Phe)) increased the mechanical stability of the complexes.

Conclusions:

• The developed methodology enables precise single-molecule mechanical measurements of ribosome-RNA interactions.
• The mechanical properties of poly(U) within ribosome complexes are comparable to single-stranded DNA.
• Initiator tRNA binding enhances the mechanical robustness of the ribosome-mRNA complex, potentially relevant for translation initiation.
• This technique opens avenues for studying other ribosome-related complexes and their mechanical responses.